Genetic engineering of microalgae for enhanced lipid production

The global demand for food, feed and fossil-based chemicals is increasing due to the continuously growing world population. Excessive and unsustainable consumption is causing anthropogenic effects resulting in high carbon emission, pollution and depletion of natural resources. Currently, the main renewable feedstocks used as alternative to petrochemical processes are derived from agricultural crops. Higher plants are a great source of oils which can be used in food, feed, paint, lighting and fuel industries. However, cultivation of crops requires vast areas of arable land, fresh water and minerals. Additionally, depletion of petrochemical resources increases the demand for plant-based fuels, creating a direct competition with food and feed production. Therefore, novel renewable and sustainable alternatives are necessary to reduce the current environmental impact of traditional industrial processes.Microalgae are considered as a promising alternative for the production of commodities. They are capable of converting sunlight, water, CO2 and nutrients into biomass and products which attract interest from pharmaceutical, cosmetic, biofuel, food and feed industries. Furthermore, microalgae can reach significantly higher biomass concentrations and product yields per hectare compared to higher plants, they do not require arable lands and some species can be grown on wastewater or seawater.Several microalgal species can accumulate large amounts of fatty acids when cultivated under stress conditions. At a cellular level, lipids such as triacylglycerols (TAGs) and polyunsaturated fatty acids (PUFAs) can serve either as structural components or storage compounds. Therefore, microalgae are a source of valuable oils that can be used directly as nutritional products or as raw material for biofuel production.Although microalgae have the potential to become a feedstock for lipid production some limitations and challenges remain. In order to achieve an economically feasible process, improvement, optimization and redesign of cultivation systems, as well as strain improvement are required. Omics and genetic tool development can accelerate the generation of strains with enhanced growth rates, photosynthetic efficiencies and lipid productivities. Therefore, genetic engineering has become a promising approach for improving strains and reducing the overall production costs.The lack of genetic tools for efficient delivery of genetic material into the cells is one of the main bottlenecks for successful transformation of microalgae. Several genetic methods have been developed such as ballistics, electroporation, Agrobacterium-mediated transformation, and agitation with glass beads. However, these techniques have been proven to work in a limited number of strains. Therefore, the development of a robust genetic toolbox that can be applied to poorly transformable strains, less well-studied or industrially relevant species is needed.Poor intracellular delivery of DNA/protein molecules into microalgae cells is caused by the structure and composition of the cell wall and membrane of each species. Additionally, successful delivery cannot be assessed by determining transformability as their functionality is not always known in the studied microorganisms. Therefore, in Chapter 2 we proposed a screening tool for the optimization of electroporation-based transformation methods. We determined cell permeability and viability using fluorescent dyes in four species with different physiological and cellular characteristics: Chlamydomonas reinhardtii, Chlorella vulgaris, Neochloris oleoabundans and Acutodesmus obliquus. We successfully delivered labelled DNA and proteins into the cells and accomplished high transformation efficiencies when using functional plasmids and applying the predicted electroporation settings. This method offers guidance to determine suitable transformation conditions for non-transformed species and increases insight on established transformation protocols.Transformation efficiency is species-dependent and some methods present several limitations: DNA fragment lengths, macromolecule size or high cell death after the treatment is applied. Therefore, a variety of methods are required to engineer a wide range of species. In Chapter 3, we presented a conjugation-based transformation technique. We successfully engineered green oleaginous microalgae strains Acutodesmus obliquus and Neochloris oleoabundans. We further attempted to deliver episomal plasmids into the cells. In our study, we did not accomplish successful rescue of episomes from transformants and we hypothesized that random integration might have occurred. This technique was proposed as an alternative transformation approach over traditional methods when successful transformation is impeded due to high cell death or physical damage and limited passage of bigger molecules through the cell wall.In Chapter 4 we enhanced TAG production in N. oleoabundans. We overexpressed genes encoding enzymes involved in the Kennedy pathway; glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase and diacylglycerol acyltransferase. Strains overexpressing single genes were able to increase total fatty acids and TAGs by 1.3- and 1.4-folds, respectively. On the other hand, simultaneous expression resulted in 1.2-fold increase. Single gene expression resulted in higher lipid titers and productivities without affecting growth rates and photosynthetic activity. Conversely, simultaneous expression resulted in lower growth rate, lower photosynthetic activity and significant changes in cell composition. Overall, our results demonstrate that heterologous gene expression can enhance lipid accumulation and can be a valid approach for the generation of strains with higher economical potential.In Chapter 5 we provided an overview of developments in genetics for the generation of microalgae strains with enhanced lipid content. We reviewed important advances in omics technology and genetic tool development as well as its application in microalgae aiming to improve carbon fluxes towards fatty acid synthesis. We provided an overview of genetic strategies including modification of fatty acid synthesis pathway, Kennedy pathway, PUFA and TAG metabolism, and modification of enzymes involved in NADPH generation and gene(s) regulation.In Chapter 6 we discussed advances in the microalgal biotechnology field including most studied strains, genetic tool development, as well as techniques for DNA delivery into microalgae cells and strain improvement approach described in this thesis. We described bottlenecks and challenges that need to be addressed in order to meet the standards of a competitive industrial strain. Development of genetic tools that can target organelles such as chloroplast or nucleus, sequence optimization for correct transcription and translation, as well of avoidance of random integration of expression systems are some of the issues that need to be tackled. Furthermore, we proposed that development of genome-editing tools is key for accurate and efficient modification of microalgae which will allow the generation of marker-free strains that can be used in industrial processes.